Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome–lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.
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The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.
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We thank V. Rawal and A.T. Veetil for valuable discussions, D. Nelson (Department of Pharmacological and Physiological Sciences, the University of Chicago) and C.A. Petersen (Department of Epidemiology, College of Public Health, University of Iowa) for providing macrophage cell lines, A. Hoffman and K. Schoenfelt (Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA) for providing primary macrophages, blood donors and K.M. Becker for help with blood draws, M. Zajac and K. Chakraborty for manuscript editing and the Integrated Light Microscopy facility at the University of Chicago. This work was supported by the University of Chicago Women’s Board, Pilot and Feasibility award from an NIDDK center grant P30DK42086 to the University of Chicago Digestive Diseases Research Core Center, R01 DK102960, MRSEC grant no. DMR-1420709, and University of Chicago start-up funds to Y.K. Pilot funding from the Biological Sciences Division to Y.K. and L.B. Y.K. is a Brain Research Foundation Fellow.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Tables 1–2, Supplementary Figures 1–19
Heat maps of phagosome acidification using z-cHOClate in G/B channel, as a function of time. Particles outside the cell have lower G/B (Blue), whereas particles inside the matured phagosome (white arrow) have higher G/B (red). Movie is played at 1 fps speed, and representative data from three independent experiments are shown.
Heat maps of HOCl production in the phagosome using z-cHOClate in R/B channel, as a function of time. Particles outside the cell show higher R/B (red), whereas particles inside the matured phagosome (white arrow) have lower R/B (blue). Movie is played at 1 fps speed, and representative data from three independent experiments are shown.
Heat maps of phagosome acidification in the phagosome using z-cHOClate in J774A.1 cells pretreated with ABAH (100 mM), visualized using the G/B channel. Particles outside the cell have lower G/B (Blue), whereas particles inside the matured phagosome (white arrow) have higher G/B (red), indicating that acidification is not hampered. Movie is played at 1 fps speed, and representative data from three independent experiments are shown.
Heat maps of HOCl production in the phagosome of J774A.1 cells pretreated with ABAH (100 mM), visualized using R/B channel. Particles outside the cell as well as particles inside the matured phagosome (white arrow) show higher R/B (red), indicating that z-cHOClate is sensing HOCl specifically in the R/B channel. Movie is played at 1 fps speed, and representative data from three independent experiments are shown.
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Thekkan, S., Jani, M.S., Cui, C. et al. A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome. Nat Chem Biol 15, 1165–1172 (2019). https://doi.org/10.1038/s41589-018-0176-3
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